New technology that enables a single cell/particle analysis based on element tags[1] is emerging in the vigorous area of proteomics and drug discovery and could be equally useful in areas of clinical and diagnostic testing. The realization that elemental analysis offers significant advantages to the field of protein quantitation has directed the development of several new methods of protein quantitation via Inductively Coupled Plasma Mass Spectrometry (ICP-MS) linked immunoassays[2-6].
Fluorescent light emitting beads (particles) are well known in a number of practical applications especially in combination with flow cytometry based methods. It was long recognized that “two or more dyes of varying proportions could be used to increase the permutation number of unique combinations of dyes in a single particle [7]”. The art is based on realization that the emission wavelengths and fluorescence intensities are useful for multi-parametric analysis of a plurality of analytes in the same sample.
Although it is feasible to have an optical emission detector capable of distinguishing a plurality of wavelengths and an excitation system with significantly broad range, it is still physically impossible to create a set of non-interfering fluorophores for a large number of unique combinations of dyes in a single particle. Typically dyes have overlapping excitation and emission spectra allowing energy transfer from the first excited dye to the next dye and through a series of dyes resulting in emission of light from the last dye in the series. Specifically for fluorescence based cytometry these limitations also include: limited dynamic range due to some spectral overlap and detector capabilities, and variability in fluorophore response (fluorophore integrity problem). In the multiplex configuration, distinguishable (meaning non-overlapping signal) fluorescent dyes are required. This results in mismatched overlap of the excitation and absorption wavelengths, with a corresponding loss in sensitivity for some of the fluorophores. More significantly, the emission spectra are not well baseline-resolved. That is, some overlap of the fluorescent signals is obtained, and this becomes important when a large signal in one channel overlaps strongly into the weaker signal channel.
On the contrary, the elemental analysis (ICP-MS, for example) has a number of unique properties that could be harnessed to create an ideal element stained particles and instrument combination for the purpose of the invention and to provide a multiplex alternative to fluorescence based methods [1].
A large number of heavy metals (as an example, the element stain, but not limited thereto) and their isotopes provide distinct signals that can be detected by MS simultaneously. Two or more elements of varying proportions could be used to increase the permutation number of unique combinations of elements in a single particle; the obtained intensity ratio vs. tag mass fingerprint could serve as a signature of the particle. Using more than two elements as staining elements yields a much larger number of detectable permutations than is feasible by fluorescently dyed particles. The method of analyzing the element-labeled particle takes advantage of the abundance sensitivity of ICP-MS, a measure of the overlap of signals of neighboring isotopes, is large (>106 for the quadrupole analyzer), and this ensures independence of the detection channels over a wide dynamic range. Further, MS is very sensitive, permitting the determination of signals at biologically significant levels. Finally, ICP-MS as a detector offers absolute quantification that is largely independent of the analyte molecular form or sample matrix. In this invention we teach, among other things, the formulation of the element stained beads and their use as a tuning standard for ICP-MS as well as functionalization of their surface to be useful as a support (for capture antibodies, for example) in bio-analytical applications.
FC-MS with element-encoded beads overcomes existing limitations of fluorescent-based suspension assays. Consider an element-encoding system for beads that employs five elements (e.g., La, Pr, Tb, Ho, Tm, all of which occur naturally as single isotopes) present at ten grades of concentration of the encoding elements, the variability will be 105−1=99,999. Elemental mass spectrometry is able to quantify up to ca. 106 grades of concentration (depending on the particular MS technique) for adjacent isotopes which translates into 1011−1 types of distinguishable “bar-coded” beads (theoretical limit). If practical isotope separation for samarium (Sm, with seven isotopes having >3% abundance) could be achieved with 99% purity, the corresponding bead-encoded system would have, assuming 10 grades of concentration, nearly ten million bead varieties (i.e., 107−1). In principle, there is no physical limit (other than MS sensitivity) to distinguishing the very large (i.e., massive) variety of element-encoded beads if MS detection is used. The polymeric element-encoded beads should not be light sensitive, will not leach out Ln ions (they are firmly encapsulated inside the fullerene), and will not require special storage conditions. In addition, there will be no background interference from sample containers or biological molecules. In one instance, we teach design a bead-based assay using polystyrene beads with embedded elemental code. The assay consists of covalently attaching a specific antibody to one type of coded bead; incubating the beads with samples containing the antigen of interest and after washing beads by low speed centrifugation probing with a reporter antibody that binds to a different epitope of the antigen and is labeled with an elemental tag. Combining several uniquely encoded beads will permit to assay multiple antigens from a single biological sample. In one instance all reporter antibodies can be labeled with the same elemental tag. The FC-MS read-out will indicate the type and quantity of antigens present in the biological sample.
Synthesis of beads intended for general bio-analytical purpose was described in the following references and, in the case of making multicolored, fluorescent beads, methods have been disclosed including: i. covalent attachment of dyes onto the surface of the particle[8], ii. internal incorporation of dyes during particle polymerization [9,10], and iii. dyeing after the particle has been already polymerized [7,11]. However, these references disclose production of fluorescent particles. Instructions for incorporation of non-fluorescent elements were not provided.
U.S. Pat. No. 6,449,562 [12] provides a general description of a multiplexed analysis of biological molecules using beads, but does not provide instructions or disclosure for providing element tagged (encoded) beads as well as no MS linked assays are disclosed.
U.S. Pat. No. 4,454,233 [13] suggests that elements embedded in “individual mobile units” could be resolved by a mass spectrometer. However, the resultant mass spectra were not provided, and synthetic strategies for “individual mobile units” with embedded elements were not presented, effectively preventing one skilled in the art to make and use such beads in mass spectrometry. As a result, no usage of the element stained beads in combination with a mass spectrometer has been practiced to date.
U.S. Pat. No. 7,225,082 provides description and preparation of rod-shaped nanoparticles. Althoght the elemental composition of the nanoparticles is varied along the length of the rod to provide viasible nanobar codes, the invention does not provide instructions or disclosure for providing element tagged (encoded) beads as well as no elemental analysis linked assays are disclosed.
Ramirez et al [14] reported miniemulsion polym-erization of butyl acrylate (BA) in water in which the BA droplets contained various lanthanides (Dy, Er, Eu, Gd, Ho, La, Pr, Yb) chelated with 2,2,6,6-tetramethyl-3,5-heptanedione. (These particular lanthanide chelates are sold commercially as NMR shift reagents.) These complexes have a low solubility in water, and the authors report preparation of poly(butyl acrylate) (PBA) particles containing a mole ratio BA/Ln complex ranging from 8.9 to 55.2. These authors did not use their metal-polymer hybrid particles as seeds for the preparation of coreshell particles, did not teach the synthesis of mixed lanthanides particles, did not encapsulate lanthanide nanoparticles into polymer particles via miniemulsion polymerization, nor did they mention any potential applications of their particles; although one skilled in the art might expect that these kind of particles could be used as a fluorophores.
Clearly, it would be an important improvement to the art to have a means of precisely element staining a particle with two or more elements premixed in a series of predetermined ratios and to have a collection of such element stained particles for use in multiplex applications. This precision in element staining process can be achieved with significantly improved ratio of the standard deviation to the mean mass spectrometer intensity of particle population in comparison with fluorescent based techniques. This fundamental difference allows incomparably more subsets or populations of non-overlapping, distinctly element stained particles to be synthesized. To ensure dramatically higher multiplex capability, it would be a further advance in the art if the methods were reproducible to within a minimal variation, to allow repeatable tuning of the elemental flow cytometer [1] across a wide variation of absolute and relative concentrations of the element stain.